Pencils and Erasers, on DNA

Summary

The author presented a novel mechanism for writing heritable programmable epigenetic memory with higher efficiency than previous dCas9-based systems.
The memory can be written using dCas9-guided DNA methylation and transcriptional suppression using the CRISPRoff system and erased using a transactivation domain as in CRISPRon.

Question

How can we “turn off” a gene?
- Change the DNA sequence?²
- Blocks transcription?³
- Introduce methyltransferase?
  - TALE⁴ ¹
  - CRISPR-dCas9⁵

Background

CRISPR-(d)Cas9⁶

CRISPR/Cas9 system introduces double-stranded break.

A “dead” Cas9 (dCas9) can be used to target specific DNA sequences without introducing double-stranded breaks.

CRISPR-dCas9-based epigenome engineering.⁵

Background

CRISPRi (CRISPR interference)³

Using dCas9 to bind to DNA and prevent TFs from binding to the promoter region and/or block RNA polymerase from transcribing the gene.

CRISPRi Overview

New Method - CRISPRoff

dCas9 guided DNA methylation and transcriptional suppression (Panel A) to achieve gene silencing that is:

durable (Panel C)
with a wide target window
independent on the presence of CpG islands.

Introduction

CRISPRoff D3A^E765A Mutant

A mutation is introduced to the Dnmt3A protein domain to prevent it from methylating DNA.
Creates a CRISPRoff system that is incapable of methylating DNA.
Used to compare methylation-dependent and methylation-independent gene silencing.

Introduction

CpG islands (CGI)

Helixitta, CC BY-SA 4.0

CpG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide (separated by a phosphate group) along its 5’ -> 3’ direction.
CpG islands are regions with a high frequency of CpG sites.
- Typically occur at or near the transcription start site of genes.
- Methylation of CpG islands is associated with gene silencing.

Introduction

Chromatin immunoprecipitation sequencing (ChIP-seq)⁷

ChIP-seq analyzes protein interaction with DNA. It is used to identify binding sites of DNA-associated proteins, such as transcription factors, histones, etc.
In this study the author used ChIP-seq to identify epigenetic modifications to Histone H3 (H3K9me3) in the genome.

Introduction

Chromatin immunoprecipitation sequencing (ChIP-seq)⁷

ChIP-seq analyzes protein interaction with DNA. It is used to identify binding sites of DNA-associated proteins, such as transcription factors, histones, etc.
In this study the author used ChIP-seq to identify epigenetic modifications to Histone H3 (H3K9me3) in the genome.

Introduction

Bisulfite sequencing PCR⁸

Detects 5-methylcytosine residues in DNA.
The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive.

The method can be combined with modern NGS technologies to provide a genome-wide view of DNA methylation patterns.

Key Findings

Epigenome editing using CRISPRoff is highly specific

Using RNAseq, near complete silencing of target genes are observed in comparison between:
- Untransfected cells (Panel A)
- Cells transfected with non-targeting sgRNA (Panel A-D)
- Cells transfected with targeting sgRNA (Panel B-D)
No significant off-target effects were observed (Panel A, E).

Transcriptome of cells with vs. without CRISPRoff (TPM = Transcripts per million)

Firstly, the author demonstrated that the CRISPRoff system is highly specific in silencing target genes.

The top panels are interesting, it basically plots the transcription level of all genes in the RNAseq. And here on panel A, the author compared completely untransfected cells vs cells that have been transfected with a non-targeting sgRNA, if we see any genes that significantly change in expression (deviate from the diagonal), it must be an off-target effect. But we can see that there are no significant changes in expression. The author also targeted three other genes, ITGB1, CD81 and CD151, and here we can see there are no outliers except in the targeting sgRNA transfected cells.

The middle panels are plotting the p values for the differential transcription of the genes. This is a more quantitative way to show the same idea, but this time by using p value it is better at comparing the significance of the changes in expression across all transcription levels.

The bottom, panel E is a manhattan plot, which is simply a plot of the p-values on the differential transcription on the y-axis across the whole genome and we can see that the data corroborates with the previous panels, that there is no significant off-target effects and the only significant change in expression is in the target genes.

Key Findings

CRISPRoff can silence genes that lack CGI annotations

CRISPRoff successfully silenced 4 out of 5 non-CGI genes. (Panel C)
CRISPRoff of genes lacking CGIs lead to a more significant decrease in cell growth compared to CRISPRoff mutants in both CGI and non-CGI genes. (Panel A)
The silencing of non-CGI genes are also methylation-dependent. (Panel E)
- Hypothesis: CALD are not silenced due to complete lack of CpG sites.

The author also demonstrated another advantage of the CRISPRoff system, that it can silence genes that lack CpG island annotations.

Let’s look at panel C first. The author selected 5 genes that lack CpG islands and tested the CRISPRoff active and mutant systems on them. The CRISPRoff system successfully silenced 4 out of 5 genes, and the mutant system did not silence any of the genes. This shows that the CRISPRoff system is capable of silencing genes that lack CpG islands in a methylation-dependent manner. Which is corroborated by the bisulfite sequencing data in panel E, where we can see that the CpG sides in non-CGI genes are more methylated after CRISPRoff treatment.

Last let’s look at panel A, this plot is showing a lot. This is a genome-wide screen of the effectiveness of CRISPRoff in silencing genes by making an sgRNA library targeting around 20k genes and measuring the growth defect of the cells. And the Y axis is the phenotype score for the active CRISPRoff system and the X axis is the phenotype score for the mutant system. So on the diagnal like is where methylation independent gene silencing would be, and down on the Y axis is where the methylation dependent gene silencing would be. And we can see that we have two observations: (1) the CRISPRoff system is more effective in silencing genes that lack CpG islands compared to the mutant system. (2) CRISPRoff achieved comparable performance between CGI and non-CGI genes.

Key Findings

CRISPRoff can silence genes that lack CGI annotations (cont’d)

CRISPRoff can silence non-CGI genes durably. (Panel F, G, I)
Silencing of non-CGI gene MYL6 is metastable. (Panel H)

Durability of gene silencing in non-CGI genes

Key Findings

CRISPRoff targeting rules

An sgRNA tiling screen was performed.
CRISPRoff has a wider target window (-500 to +500 bp from TSS¹) compared to CRISPRi (-75 to +75 bp from TSS). (Panel E, H)
Active sgRNAs are not evenly distributed but instead appear in a periodic pattern. (Panel D, I)
- Hypothesis: The periodic pattern is due to the nucleosome positioning.

Next topic is the sgRNA design rules for the CRISPRoff system. Similar to the CRISPRi system, the CRISPRoff system requires a well-designed sgRNA to achieve efficient gene silencing. The author performed an sgRNA tiling screen (which is basically a library of all possible combinations) to determine the optimal sgRNA design rules for the CRISPRoff system. On panel E and H we observe that CRISPRoff has a much wider target window compared to CRISPRi, from +-75bp to +-500bp from the transcription start site. This is a significant improvement in the CRISPRoff system, as it allows for more flexibility in the design of the sgRNA.

The author also observed that the distribution of active sgRNAs are not a simple V-shaped distribution, but instead appear in a periodic pattern. This is an interesting observation that the author hypothesized is due to the nucleosome positioning.

Key Findings

CRISPRon can turn on silenced genes

Two constructs are used to create CRISPRon (Panel F).
- The first is a dCas9-TET1 fusion protein (TETv4) that demethylates DNA. (Panel A).
- The second is a sequence of transactivator domains fused with a MS2 coat protein (MCP) (Panel E). MS2 coat protein binds to the MS2 loops on the sgRNA achieving recruitment of the transactivator domains to the target gene (Panel F).

Key Findings

CRISPRon can turn on silenced genes (cont’d)

Reactivation of genes is DNA-demethylation dependent (panel G, H, top).
TETv4 alone is not effective in reactivating previously silenced genes (Panel G, right).
In summary, both TETv4 and transactivator are necessary for most effective reactivation of genes.

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Next Steps

Optimization of the sgRNA design rules for CRISPRoff.
Why are some genes not durably silenced?
More research into the functional effects of artifically introduced DNA methylation.

Future Directions of CRISPRon

What is the efficacy of CRISPRon on genes that are naturally methylated?
- Design sgRNAs to target several genes that are naturally methylated. Use a library tiling screen to help determine the optimal sgRNA design.
- Use bisulfite sequencing to determine the methylation status of the genes, before and after CRISPRon, with and without using transactivators.
- Measure the differential transcription of the genes using RNAseq.
- Measure differential protein production using a tagged protein and Western blotting or a fluorescent protein and flow cytometry.

References

(1)

Nuñez, J. K.; Chen, J.; Pommier, G. C.; Cogan, J. Z.; Replogle, J. M.; Adriaens, C.; Ramadoss, G. N.; Shi, Q.; Hung, K. L.; Samelson, A. J.; Pogson, A. N.; Kim, J. Y. S.; Chung, A.; Leonetti, M. D.; Chang, H. Y.; Kampmann, M.; Bernstein, B. E.; Hovestadt, V.; Gilbert, L. A.; Weissman, J. S. Genome-Wide Programmable Transcriptional Memory by CRISPR-Based Epigenome Editing. Cell 2021, 184 (9), 2503–2519.e17. https://doi.org/10.1016/j.cell.2021.03.025.

(2)

Dalvie, N. C.; Lorgeree, T.; Biedermann, A. M.; Love, K. R.; Love, J. C. Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination. ACS Synthetic Biology 2022, 11 (1), 497–501. https://doi.org/10.1021/acssynbio.1c00194.

(3)

Larson, M. H.; Gilbert, L. A.; Wang, X.; Lim, W. A.; Weissman, J. S.; Qi, L. S. CRISPR Interference (CRISPRi) for Sequence-Specific Control of Gene Expression. Nature Protocols 2013, 8 (11), 2180–2196. https://doi.org/10.1038/nprot.2013.132.

(4)

Bernstein, D. L.; Le Lay, J. E.; Ruano, E. G.; Kaestner, K. H. TALE-Mediated Epigenetic Suppression of CDKN2A Increases Replication in Human Fibroblasts. The Journal of Clinical Investigation 2015, 125 (5), 1998–2006. https://doi.org/10.1172/JCI77321.

(5)

Xu, X.; Qi, L. S. A CRISPR–dCas Toolbox for Genetic Engineering and Synthetic Biology. Journal of Molecular Biology 2019, 431 (1), 34–47. https://doi.org/10.1016/j.jmb.2018.06.037.

(6)

Ran, F. A.; Hsu, P. D.; Wright, J.; Agarwala, V.; Scott, D. A.; Zhang, F. Genome Engineering Using the CRISPR-Cas9 System. Nature Protocols 2013, 8 (11), 2281–2308. https://doi.org/10.1038/nprot.2013.143.

(7)

Johnson, D. S.; Mortazavi, A.; Myers, R. M.; Wold, B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (New York, N.Y.) 2007, 316 (5830), 1497–1502. https://doi.org/10.1126/science.1141319.

(8)

Frommer, M.; McDonald, L. E.; Millar, D. S.; Collis, C. M.; Watt, F.; Grigg, G. W.; Molloy, P. L.; Paul, C. L. A Genomic Sequencing Protocol That Yields a Positive Display of 5-Methylcytosine Residues in Individual DNA Strands. Proceedings of the National Academy of Sciences of the United States of America 1992, 89 (5), 1827–1831.

Pencils and Erasers, on DNA

Summary

Question

Background

CRISPR-(d)Cas96

Background

CRISPRi (CRISPR interference)3

New Method - CRISPRoff

Introduction

CRISPRoff D3AE765A Mutant

Introduction

CpG islands (CGI)

Introduction

Chromatin immunoprecipitation sequencing (ChIP-seq)7

Introduction

Chromatin immunoprecipitation sequencing (ChIP-seq)7

Introduction

Bisulfite sequencing PCR8

Key Findings

Epigenome editing using CRISPRoff is highly specific

Key Findings

CRISPRoff can silence genes that lack CGI annotations

Key Findings

CRISPRoff can silence genes that lack CGI annotations (cont’d)

Key Findings

CRISPRoff targeting rules

Key Findings

CRISPRon can turn on silenced genes

Key Findings

CRISPRon can turn on silenced genes (cont’d)

Next Steps

Future Directions of CRISPRon

References

CRISPR-(d)Cas9⁶

CRISPRi (CRISPR interference)³

CRISPRoff D3A^E765A Mutant

Chromatin immunoprecipitation sequencing (ChIP-seq)⁷

Chromatin immunoprecipitation sequencing (ChIP-seq)⁷

Bisulfite sequencing PCR⁸